"Ketocarotenoid" is a general term for keto group-containing carotenoid pigments. Carotenoids are synthesized from mevalonic acid as a starting substance via isoprenoid basic biosynthesis pathway which shares an initial part with the synthesis pathway for steroids and other isoprenoids (see FIG. 6). Isopentenyl pyrophosphate (IPP) with 5 carbon atoms, which is a basic unit, generated from the isoprenoid basic biosynthesis pathway condenses with its isomer dimethylallyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP) with 10 carbon atoms and, in addition, IPP condenses to produce farnesyl pyrophosphate (FPP) with 15 carbon atoms. FPP produces geranylgeranyl pyrophosphate (GGPP) with 20 carbon atoms by condensing with IPP again. Then, GGPPs condense with each other to produce colorless phytoene which is the initial carotenoid. Through a series of unsaturated reactions, phytoene is converted to phytofluene, .zeta.-carotene, neurosporene and finally to lycopene. Subsequently, lycopene is converted by a cyclization reaction to a .beta.-carotene containing two .beta.-ionone rings. Finally, it is believed that a keto-groups, a hydroxyl group, etc. are introduced into the .beta.-carotene to thereby synthesize astaxanthin, zeaxanthin and the like (see Britton, G., "Biosynthesis of Carotenoids", Plant Pigments, Goodwin, T. W (ed.), London, Academic Press, 1988, pp. 133-182).
Recently, the present inventors have cloned a group of carotenoid biosynthesis genes of the non-photosynthetic bacterium Erwinia uredovora present in plant from the genomic DNA library in E. coli using its yellow color formation as an indicator. Further, by expressing a various combinations of these genes in microorganisms such as E. coli, the inventors has made it possible to produce in microorganisms such as E. coli phytoene, lycopene, .beta.-carotene and zeaxanthin which is a yellow carotenoid pigment wherein a hydroxyl group has been introduced into .beta.-carotene (see FIG. 7) (Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y., Nakamura, K., and Harashima, K., "Elucidation of the Erwinia uredovora Carotenoid Biosynthetic Pathway by Functional Analysis of Gene Products Expressed in Escherichia coli", J. Bacteriol., 172, pp. 6704-6712, 1990; Misawa, N., Yamano, S, Ikenaga, H., "Production of .beta.carotene in Zymomonas mobilis and Agrobacterium tumefaciens by Introduction of the Biosynthesis Genes from Erwinia uredovora", Appl. Environ. Microbiol., 57, pp. 1847-1849, 1991; and Japanese Unexamined Patent Publication No. 3-58786).
On the other hand, astaxanthin which is a red ketokarotenoid is a representative animal carotenoid widely present in marine organisms, e.g. red fishes such as sea bream and salmon, and crustaceans such as crab and shrimp. Since animals generally cannot biosynthesize carotenoids, they have to take in from outside those catotenoids synthesized by microorganisms or plants. For this reason, astaxanthin has been widely used for the purpose of red color enhancing for cultured fishes and shellfishes such as sea bream, salmon and shrimp.
Astaxanthin is also used as a coloring agent for foods. Furthermore, astaxanthin is attracting attention as an antioxidant to remove activated oxygen generated in a body which is causative of a cancer (see Takao Matuno and Wataru Inui, "Physiological Functions and Biological Activities of Carotenoids in Animals", KAGAKU TO SEIBUTU (Chemistry and Organisms), 28, pp. 219-227, 1990).
As sources of astaxanthin supply, there are known crustaceans such as antarctic krill, a culture of the yeast Phaffia, a culture of the green alga Haematococcus and compounds which are obtained by organic synthesis. However, when crustaceans such as antarctic krill are used, it is difficult to separate astaxanthin from various contaminants, such as lipids, in a recovery and extraction process, which requires a great labor and cost. When a culture of the yeast Phaffia is used, the recovery and extraction of astaxanthin also requires a great cost since its cell wall is rigid and yet the production level of astaxanthin is low. In the case of using a culture of the green alga Haematococcus, it is necessary to supply to the alga during its cultivation some light which is essential for astaxanthin synthesis. Therefore, appropriate conditions on a location for taking sun light in or cultivation facilities capable of supplying artificial light are required. In addition, it is difficult to separate the produced astaxanthin from mixed up chlorophyl and by-products (fatty acid esters). For these reasons, it has been true that the organism-derived astaxanthin described above cannot compete with those obtained by organic synthesis in cost. However, considering that astaxanthin is used as feed for fishes and shellfishes and as a food additive, an astaxanthin prepared by organic synthesis has some problems with respect to by-products produced in the reaction and yet such an astaxanthin is against the consumers' liking for natural products.
Under circumstances, the development of a method for producing an organism-derived cheap astaxanthin which is safe and can meet the consumers' liking for natural products is desired.
Then, it is believed that the acquisition of a group of genes involved in the biosynthesis of astaxanthin would be very useful, because it is possible to render an optimal microorganism with respect of safety as a food and a potential ability to produce astaxanthin, regardless of whether it has an ability to produce astaxanthin or not, the production ability by introducing into the microorganism the group of astaxanthin synthesis genes and expressing them. In this case, there will occur no problem of the mixing of by-products. In addition, by using techniques of the highly advanced genetic engineering, it will not be difficult to increase the amount of astaxanthin production to a level which exceeds the production amount by organic synthesis. As described above, a group of genes to synthesize up to zeaxanthin have already been obtained by the present inventors from the non-photosynthetic bacterium Erwinia uredovora. However, no one has succeeded in obtaining the gene coding for a keto group-introducing enzyme that is necessary for synthesizing astaxanthin, though a number of attempts have been made in many research institute because of the industrial utility of astaxanthin as described above. As to the reasons, it is considered that enzymes located downstream and involved in carotenoid biosynthesis, such as a keto group-introducing enzyme, are membrane proteins and that the purification and measurement of activity of those enzymes have been impossible; therefore, there has been no finding about those enzymes. In particular, as to a keto group-introducing enzyme, not only findings about the enzyme itself but also findings about the gene coding for the enzyme have not been reported at all. Therefore, to date, it has been impossible to produce astaxanthin in a microorganism or the like by using genetic engineering techniques.